Influenza is a continuing worldwide public health problem associated with significant morbidity and mortality. Seasonal influenza epidemics affect about 10% of the world's population, and annual estimates of mortality range from 250,000 ? 500,000 deaths. Due to constant antigenic drift, the approved seasonal influenza vaccine is variably effective from year to year, and a universal vaccine is still a long way off. Two classes of antivirals have been developed, the adamantanes, which target the viral M2 protein, and the viral neuraminidase (NA) inhibitors. However, drug resistance has abolished clinical adamantane use, and there is a growing problem of resistance to oseltamivir, the most widely prescribed of the NA inhibitors. Because of drug resistance issues, lack of a universal vaccine, and the threat of future pandemics, there is a clear and pressing need for development of novel anti-influenza therapeutics. Influenza virus is an enveloped, negative strand RNA virus whose genome is copied in the nucleus of infected cells. The viral nucleoprotein NP plays critical roles in RNA packaging, transcription of the genome to the positive sense species, and RNA replication to negative sense genomes for new virus production. For these reasons NP is an attractive drug target. Alexander BioDiscoveries, LLC has identified novel, specific, potent inhibitors of NP using an innovative, yeast- based antiviral drug discovery technology. These inhibitors bind directly to NP and also prevent its accumulation in the nucleus of infected cells, accounting for its potent antiviral activity. A one-year Phase I feasibility study is proposed to establish robust SAR that will lay the groundwork for a future Phase II study directed at lead optimization, PK and in vivo efficacy studies. In Specific Aim 1, a comprehensive medicinal chemistry approach will be undertaken to improve potency, establish SAR and ensure drug-like properties. Two distinct starting points have been chosen, each based on compounds with excellent antiviral activity in cell culture and minimal cytotoxicity. Combinatorial and discrete syntheses will be conducted to generate numerous analogs for in vitro binding studies and antiviral testing. For Specific Aim 2, a combination of antiviral, in vitro binding, cytotoxicity, and NP nuclear localization assays will be used to assess activity of analogs generated in Aim 1. Selected compounds will also be compared with oseltamivir and in addition be used in combination with oseltamivir. Broad-spectrum activity will be addressed using a variety of recent H1N1 and H3N2 seasonal strains, including oseltamivir-resistant and adamantane-resistant strains. Selected compounds will be used to select viral escape mutants in order to characterize the genetics of drug resistance, if it exists, and the fitness of resistant viral variants.